大野論文正誤表

Figure S1. DNA quality control. TapeStation profiles of gDNA isolated from FF and matching FFPE block tumor tissues from 5 lung ADC patients. In each profile, the DIN, indicative of gDNA degradation status, is also displayed (numerical assessment ranges from 10 for undamaged gDNA, to 1 for highly fragmented gDNA) (a). The Table reports the gDNA concentration (ng/ul) assessed by NanoDrop, Qubit, and TapeStation, and purity (260/280 and 260/230) (b). Additionally, AYR and DIN parameters, indicative of FFPE gDNA fragmentation status, evaluated by a multiple PCR assay and TapeStation respectively, are reported. Image of agarose gel 1 % shows the gDNA smears indicative of the different degradation status of FF and FFPE gDNAs (c). Figure S2. The workflow illustrates samples processing and WES data analysis for both exome enrichment platforms. (PDF 187 kb

Supplementary audio files for ‘Chromas from chromatin: Sonification of the epigenome’

Supplementary audio S1.1: Audio track extracted from region chr17:7531293-7631293<br>Supplementary audio S2.1: Audio track extracted from region chr4:9734445-9834445<br>Supplementary audio S3.1: Audio track extracted from region chr8:145689938-145789938<br>Supplementary audio S4.1: Audio track extracted from region chr2:25460307-25560307<br>Supplementary audio S5.1: Audio track extracted from region chrX:44802134-44902134<br>Supplementary audio S6.1: Audio track extracted from region chr15:44957020-45057020<br>Supplementary audio S7.1: Audio track extracted from region chr13:30986479-31086479<br>Supplementary audio S8.1: Audio track extracted from region chr10:89625864-89725864<br>Supplementary audio S9.1: Audio track extracted from region chr16:10944948-11044948<br>Supplementary audio S10.1: Audio track extracted from region chr8:128700997-128800997<br>Supplementary audio S1.2: Randomized audio track extracted from region chr17:7531293-7631293<br>Supplementary audio S2.2: Randomized audio track extracted from region chr4:9734445-9834445<br>Supplementary audio S3.2: Randomized audio track extracted from region chr8:145689938-145789938<br>Supplementary audio S4.2: Randomized audio track extracted from region chr2:25460307-25560307<br>Supplementary audio S5.2: Randomized audio track extracted from region chrX:44802134-44902134<br>Supplementary audio S6.2: Randomized audio track extracted from region chr15:44957020-45057020<br>Supplementary audio S7.2: Randomized audio track extracted from region chr13:30986479-31086479<br>Supplementary audio S8.2: Randomized audio track extracted from region chr10:89625864-89725864<br>Supplementary audio S9.2: Randomized audio track extracted from region chr16:10944948-11044948<br>Supplementary audio S10.2: Randomized audio track extracted from region chr8:128700997-12880099

Mutant uromodulin expression leads to altered homeostasis of the endoplasmic reticulum and activates the unfolded protein response

<div><p>Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle’s loop (TAL) and it plays key roles in kidney function and disease. Mutations in <i>UMOD</i>, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-<i>UMOD</i>), characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of <i>UMOD</i> mutations, retention in the endoplasmic reticulum (ER), is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-<i>UMOD</i> pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells) of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR), and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-<i>UMOD</i> pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and associated protein degradation pathways.</p></div

Analysis of UPR induction in mTAL cells expressing different uromodulin mutants.

<p>(A) <i>Bip</i> and spliced <i>Xbp1</i> expression assessed by real time RT-qPCR. Expression is normalised to Hprt1. *P<0.05, **P<0.01 (Student t test <i>vs</i> GFP and WT) (n = 6 independent experiments). (B) Western blot analysis of PERK in mTAL cells expressing mutant uromodulin isoforms. None of the mutant isoforms is inducing PERK phosphorylation, seen as a shift in protein migration, as observed upon tunicamycin treatment (2 μg/mL for 14 h). (C) ATF6 activity as assessed by use of an ATF6 reporter construct. No ATF6 activation is observed in mutant uromodulin expressing cells. WT cells treated with tunicamycin are shown as a positive control *P<0.05 (Student t test <i>vs</i> WT) (n = 6 independent experiments).</p

UPR induction in mTAL cells expressing wild type or C150S mutant uromodulin isoform.

<p>(A) <i>Bip</i> and spliced Xbp1 (<i>Xbp1s</i>) expression assessed by real-time RT-qPCR. Expression is normalised to <i>Hprt1</i> (n = 5 independent experiments) (left panel). Western blot analysis showing increased Bip protein levels in mTAL cells expressing mutant uromodulin (n = 3 independent experiments) (right panel). *P<0.05 (mutant <i>vs</i> wild type, Student t test). (B) Western blot analysis of PERK in mTAL cells expressing uromodulin at baseline and after incubation with tunicamycin (2 μg/mL for 14h). A shift in PERK migration is observed after treatment with tunicamycin, but not at baseline (left panel). Western blot performed with an antibody specific for the phosphorylated (Thr980) form of PERK shows the presence of the phosphorylated protein only in tunicamycin-treated cells (right panel). (C) ATF4 activity, assessed through the use of a luciferase-based, ATF4 reporter construct, is equally negligible in cells expressing wild type or C150S uromodulin, while it is evident in all cells upon thapsigargin treatment (100 nM for 14 h) ***P<0.001 (control <i>vs</i> thapsigargin, Student t test) (n = 6). (D) ATF6 activation assessed through the use of a luciferase-based, ATF6 reporter construct. No ATF6 activation is observed in mutant uromodulin expressing cells. Activation can be observed in all cell lines upon treatment with tunicamycin. ***P<0.005 (control <i>vs</i> tunicamycin, Student t test) (n = 8).</p

Stability of wild type and C150S mutant uromodulin in mTAL cells.

<p>(A) Pulse-chase experiment showing maturation of wild type and C150S uromodulin isoforms in mTAL cells. Wild type uromodulin is completely matured into the Golgi-type glycosylated form (white arrow) after 4 hours of chase, while the mutant one shows mainly the ER-type glycosylated form (black arrow) even after 12 hours. (B) Pulse-chase experiment showing mutant uromodulin stability in presence of proteasome (MG132) or autophagy (bafilomycin) inhibitors. Mutant uromodulin is stabilised by treatment with MG132 suggesting the involvement of the proteasome for its degradation. *P<0.05, **P<0.01, ***P<0.005 (Student t test) (n = 4 independent experiments). (C) Western blot analysis showing increased co-immunoprecipitation of ER chaperones calnexin, PDI and BiP with mutant uromodulin relative to wild type one.</p

Transcriptome analysis in mTAL cells expressing wild type or C150S mutant uromodulin.

<p>(A) Heat map showing differentially expressed genes in mTAL cells expressing C150S uromodulin compared to wild type ones. Cut off: fold change > 1.5; P adjusted < 0.05. (B) STRING analysis showing networks formed by proteins encoded by up-regulated genes in mTAL cells expressing C150S uromodulin. Edges represent protein-protein association (physical or functional); their thickness is proportional to confidence. (C) Same analysis as in panel B for down-regulated genes. The proteins encoded by these genes are not forming relevant networks.</p

UPR induction in MDCK cells expressing wild type or C150S uromodulin isoforms.

<p>(A) <i>BIP</i> and <i>XBP1S</i> expression assessed by real-time RT-qPCR. Expression is normalised to <i>HPRT1</i>. *P<0.05, **P<0.01 (Student t test) (n = 5 independent experiments). (B) Western blot analysis of PERK in MDCK cells expressing wild type or C150S mutant uromodulin. A shift in PERK migration is seen upon tunicamycin treatment (2 μg/mL for 14 h), but not at baseline. (C) ATF6 activation assessed through the use of an ATF6 reporter construct. No ATF6 activation is observed in mutant uromodulin expressing cells. Activation can be observed in all cell lines upon tunicamycin treatment. ***P<0.005 (control <i>vs</i> tunicamycin-treated, Student t test) (n = 6 independent experiments).</p

Characterisation of mTAL cells expressing wild type or mutant uromodulin isoforms.

<p>(A) Live imaging showing GFP signal in mTAL cells expressing GFP-tagged WT or C150S mutant uromodulin isoform. Bar = 40 μm. (B) Uromodulin expression assessed by real-time RT-qPCR. Expression is normalised to <i>Hprt1</i>. Cells expressing GFP alone are shown as negative control (n = 5 independent experiments) (C) Western-blot analysis of mTAL cells expressing WT or C150S mutant uromodulin isoform. * indicates the ER glycosylated form that is Endo H sensitive (see panel below). (D) Immunofluorescence analysis of mTAL cells expressing GFP-tagged uromodulin isoforms. GFP signal is shown in green. Calreticulin, used as an ER marker, is shown in red. Merged pictures show ER localisation of mutant uromodulin while the wild type protein is trafficked to the membrane. Bar = 40 μm.</p

Upregulated pathways in mTAL cells expressing C150S uromodulin compared to wild type ones.

<p>Upregulated pathways in mTAL cells expressing C150S uromodulin compared to wild type ones.</p